RNA Vectors with Hairpin-Like Inserts

This application claims priority to U.S. Provisional Patent Appln. Ser. No. 63/338,290 (filed on May 4, 2022; pending), which application is hereby incorporated by reference herein in its entirety.

This invention was made with government support under 20207002933198 awarded by the United States Department of Agriculture (USDA) and under MCB2034359 awarded by the National Science Foundation (NSF). The United States government has certain rights in this invention.

This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in computer-readable media (file name: 30059-P72448US04_93707816_SequenceListing, created on Jun. 13, 2025, and having a size of 217,368 bytes), which file is herein incorporated by reference in its entirety. The sequences in the listing are RNA sequences, but use “t” to denote uracil pursuant to WIPO st.26 standards.

The present disclosure relates to a vector having an exogenous RNA segment. The vector may be suitable for introducing a therapeutic agent, such as a peptide, a protein or a small RNA, into a host. In some examples the host is a plant, wherein movement of the vector (but not necessarily the agent) is optionally limited to the phloem and the agent may be targeted to control or manage a plant disease or condition.

Both general and highly targeted anti-microbial agents have been developed for animals (e.g., humans) whose circulatory systems provide a delivery system for widespread application throughout the animal. In contrast, much less research has been conducted to develop general or targeted therapeutic agents for non-genetically modified plants since lack of a simplified circulatory system complicates delivery throughout the host plant. This is especially problematic in large, long-lived trees (e.g., citrus), where injection of anti-microbial agents may be rapidly diluted. As a result, few solutions exist for treating systemic plant infections or conditions beyond external application of pesticides, e.g., to control the pathogen's vector during the growing season, foliar applications to strengthen a plant's health in general, or expensive, short-duration injection of agents targeting the pathogen or vector.

Plant industries are at substantial risk from various pathogens. Particularly concerning are diseases and conditions affecting the citrus industry. Huanglongbing (HLB), also known as Citrus Greening, is the most serious citrus disease globally. HLB is associated with three species of the bacterium Candidatusspp. (, and) and is transmitted by two psyllid species, Asian citrus psyllid (ACP) () and African citrus psyllid (, Del Guercio). HLB is graft-transmissible and spreads naturally when a bacteria-containing psyllid feeds on a citrus tree and deposits the pathogenic bacteria into the phloem where the bacteria reproduce. The infected tree reacts by producing excessive callose in its phloem in order to isolate the bacteria, which restricts the flow of photoassimilates and can ultimately kill the tree. Once a tree is infected, there is no cure. While the diseased fruit pose no health threat to humans, HLB has devastated millions of acres of citrus groves throughout the world. In the United States alone, ACP and CL(CLas) have decimated the Florida citrus industry, causing billions of dollars of crop losses within a very short time span. Moreover, HLB has spread into every citrus producing region in the United States. Most infected trees die within a few years after infection, and fruit develops misshapen and off flavored and thus is unsuitable for consumption. According to the United States Department of Agriculture (USDA), the entire citrus industry is at substantial risk.

Consideration of plant physiology aids in the development and implementation of strategies for managing plant diseases and conditions. The vascular system of plants is the key conduit for the movement of sugars and amino acids, as well as signaling molecules such as small ribonucleic acids (RNAs), messenger RNAs (mRNAs), proteins, peptides and hormones, which are required for a large number of developmental processes and responses to biotic and abiotic stress () (Lee, J. Y. and Frank, M. (2018),, Curr Opin Plant Biol 43:119-124; Tugeon, R. and Wolf, S. (2009),, Ann Rev Plant Biol 60:207-221). Many RNAs comprise a portion of these transtiting molecules, and thousands of companion cell mRNAs can be isolated from neighboring enucleated sieve elements, where they are transported bidirectionally by osmotically generated hydrostatic pressure from source (sugar generating) tissue to sink (sugar utilizing) tissue such as roots and shoot tips (Folimonova, S. Y. and Tilsner, J. (2018),, Curr Opin Plant Biol 43:82-88; Ham, B. K. and Lucas, W. J. (2017),-, Annual Rev Plant Biol 68:173-195). As much as 50% of the companion cell transcriptome is believed to be engaged in facilitating the movement of such molecules (Kim, G. et al. (2014),-, Science 345:808-811; Thieme, C. J. et al. (2015),, Nature Plants 1(4):15025; Yang, Y. et al. (2015),, BMC Plant Biol 15, 251), which raises various questions with regard to how and why such a substantial subset of mRNAs are moving long-distances. For example, how selective is the process of RNA movement? If there is selection, how is it facilitated? Are transiting RNAs modified (e.g., methylated)? Are transiting RNAs found in any particular subcellular location before exiting into the SE? Are there “address codes” for transiting RNAs to particular locations? Are transiting RNAs bound by specific proteins and are there specific interacting sequences? How much of the flow of mRNAs is biologically meaningful and how much is non-selective, since sink cells are presumably capable of transcribing the same mRNAs?

Confusion in the mRNA movement literature is pervasive. Some studies have indicated that the major determinant of RNA mobility is their abundance in companion cells (Kim, G. et al. (2014),-, Science 345:808-811; Thieme, C. J. et al. (2015),, Nature Plants 1(4):15025; Yang, Y. et al. (2015),, BMC Plant Biol 15, 251). Mathematical modeling has been used to propose a non-selective, Brownian diffusion model for mRNA movement based mainly on their abundance, with half-life and transcript length also playing roles (Calderwood, A. et al. (2016),, Plant Cell 28:610-615). However, other studies have reached opposing conclusions, finding that mRNA abundance in companion cells does not correlate with movement (Xia, C. et al. (2018),-, Plant Physiol 177:745-758). In addition, while it is generally assumed that the phloem does not contain RNases that target the transiting RNAs (Morris, R. J. (2018),-, Curr Opin Plant Biol 43:1-7), Xia et al. also found that most mobile mRNAs are degraded and never reach the root or upper stem. Other studies found that the presence of a predicted tRNA-like structure is associated with over 11% of mobile mRNAs (Zhang, W. N. et al. (2016),-, Plant Cell 28:1237-1249), suggesting that mobile mRNAs might harbor specific “zip-codes”. However, other abundant mRNAs containing similar tRNA-like motifs were not mobile (Xia, C. et al. (2018),-, Plant Physiol 177:745-758). Thus, prior studies have failed to identify and develop a model system consisting of a highly abundant, mobile RNA whose movement is traceable in living tissue under different cellular conditions.

Plant viruses, many of which move through the plant as a ribonucleoprotein complex (vRNP), have evolved to use the same pathway as used by mobile endogenous RNAs. Plant viruses can accumulate in substantial amounts, and most initiate infection in epidermal or mesophyll cells and then move cell-to-cell through highly selective intercellular connectors called plasmodesmata, which allow for continuity between the cytoplasm of neighboring cells (; see also Lee, J. Y. and Frank, M. (2018),, Curr Opin Plant Biol 43:119-124; Schoelz, J. E. et al. (2011),, Mol Plant 4:813-831). Long-distance systemic movement (leaf-to-leaf) requires that the virus enters companion cells, where replication takes place, followed by progeny exit into sieve elements by transiting through the specialized, branched plasmodesmata that connect companion cells and sieve elements. Once tubular sieve elements are reached, viruses move passively with the phloem photoassimilate stream and establish systemic infections upon exiting (Folimonova, S. Y. and Tilsner, J. (2018),, Curr Opin Plant Biol 43:82-88).

For viruses that transit through the phloem as viral nucleoproteins (vRNPs), movement is similar to that of host mRNAs. All plant viruses encode at least one movement protein necessary for movement, which binds to viral RNA and also dilates plasmodesmata. Thus, host mRNA movement also likely requires similar host-encoded movement proteins. Viral movement proteins are non-specific RNA binding proteins. However, questions remain with regard to how vRNPs load into the phloem and unload in distal tissues, although reprograming companion cell gene expression may be required (Collum, T. D. et al. (2016),-, Proc Natl Acad Sci USA 113:E2740-E2749). If mRNA trafficking is so widespread and non-specific, it has remained unclear why RNA viruses require their own encoded movement proteins. Some researchers have suggested that RNA viruses require movement proteins if they move as preformed replication complexes that include a large RNA-dependent RNA polymerase (Heinlein, M. (2015),, Virology 479:657-671), which is beyond the size-exclusion limit (˜70 kDa) of companion cell plasmodesmata. It has also remained unclear why and how some viruses are phloem-limited. For example, phloem-limited closteroviruses have at least 3 movement proteins, and phloem-limitation can be relieved by over-expressing the silencing suppressor and downregulating host defenses (Folimonova, S. Y. and Tilsner, J. (2018),, Curr Opin Plant Biol 43:82-88), suggesting that phloem-limitation is a complex process for some viruses. Phloem-limitation can also be an active process (as opposed to lack of a cell-to-cell movement protein). For example, altering a domain of the Potato leaf role virus movement protein conferred the ability to exit the phloem (Bendix, C., and Lewis, J. D. (2018),-, Mol Plant Path 19:238-254).

A direct connection between host movement of mRNAs and vRNP movement was established when the origin of plant virus movement proteins was solved. A pumpkin protein (RPB50) related to the Cucumber mosaic virus movement protein was discovered that was capable of transporting its own mRNA, as well as other mRNAs, into the phloem (Xoconostle-Cazares, B. et al. (1999),, Science (New York, NY) 283:94-98; Ham, B. K. et al. (2009),50-, Plant Cell 21:197-215). A complex population of these endogenous movement proteins, known as non-cell-autonomous proteins (NCAPs), have been proposed as being responsible for the long-distance phloem trafficking of mRNAs (Gaupels, F. et al. (2008),, New Phytol 178:634-646; Gomez, G. et al. (2005),--, Plant J 41:107-116; Kim, M. et al. (2001),-, Science (New York, NY) 293:287-289; Pallas, V. and Gomez, G. (2013),--, Front Plant Sci 4:130; Yoo, B. C. et al. (2004),, Plant Cell 16:1979-2000).

Since their discovery (Deom, C. M. et al. (1987),30-, Science (New York, NY) 237:389-394), a number of viral movement proteins have been identified that are responsible for intracellular trafficking of vRNPs to the plasmodesmata, as well as for cell-to-cell and long-distance movement (Tilsner, J. (2014),, J Microscopy 258(1):1-5). For some viruses (e.g., umbraviruses), cell-to-cell and long-distance movement is associated with multiple movement proteins (Ryabov, E. V. et al. (2001),-and, Virology 288:391-400). For example, closteroviruses such as Citrus tristeza virus contain three movement proteins. However, for many viruses, all movement activities are thought to be associated with a single movement protein.

Delivering engineered therapeutic agents into plants for combating diseases, insects or other adverse conditions (e.g., HLB and/or the carrier insects) using virus vectors is an established means of introducing traits such as resistance to pathogens or other desired properties into plants for research purposes. Various methods of providing vectors to plants are known in the art. This is often achieved by delivery of the virus vector into a plant cell's nucleus by Agrobacteria tumefactions-mediated “agroinfiltration,” which may result in a modification of that cell's genome, or by delivering the virus vector directly into a cell's cytoplasm, which results in infection without a requirement for genomic modification. In the case of agroinfiltration of RNA viruses, the cDNA of the viral genome is incorporated into the T-DNA, which Agrobacteria delivers into the plants. Such T-DNA includes further regulatory DNA components (e.g., promoter for RNA polymerase), which allow for transcription of the viral genome within plant cells. The incorporated virus, containing therapeutic DNA inserts, is transcribed into RNA within the plant cells, after which the virus behaves like a normal RNA virus (amplification and movement).

Thus, to act as an effective vector, a virus should be engineered to accept inserts without disabling its functionality and to ensure that the engineered virus is able to accumulate systemically in the host to a level sufficient to deliver and in some cases express the insert(s). These inserts, whether having open reading frames (ORFs) that will be translated into proteins or non-coding RNAs that will be used for a beneficial function, should be delivered into the targeted tissue in a manner that is effective and sufficiently non-toxic to the host or to any downstream consumption of the host or the environment. However, only a limited number of viral vectors exist that meet the above criteria and these are available for only certain plants (e.g., citrus tristeza virus for citrus). Unfortunately, there is either no known suitable viral vector, or only suboptimal viral vectors, for most plants, particularly for long lived trees and vines. Moreover, maintaining stability of short sequences inserted into a vector has raised numerous challenges. As apparent from prior research on virus vectors, the ability to stabilize inserted sequences utilizing conventional methodologies has not been successful, particularly for long periods (e.g., months or years). Conventional vectors quickly evolve or mutate, discarding all or part of the inserted sequences since the native virus (without inserts) is generally more fit than viral vectors containing such inserts.

Thus, the ability to implement RNA or DNA therapies on abroad basis is substantially limited with existing technologies. Over 1,000 plant viruses have been identified with many plants subject to infection by multiple viruses. For example, citrus trees are subject to citrus leaf blotch virus, citrus leaf rugose virus, citrus leprosis virus C, citrus psorosis virus, citrus sudden death-associated virus, citrus tristeza virus (CTV), citrus variegation virus, citrus vein enation virus and citrus yellow mosaic virus, among others. However, CTV, the causal agent of catastrophic citrus diseases such as quick decline and stem pitting, is currently the only virus that has been developed as a vector for delivering agents into citrus phloem.

CTV is a member of the genus Closterovirus. It has a flexuous rod-shaped virion composed of two capsid proteins with dimensions of 2000 nm long and 12 nm in diameter. With a genome of over 19 kb, CTV (and other Closteroviruses) are the largest known RNA viruses that infect plants. It is a virulent pathogen that is responsible for killing, or rendering useless, millions of citrus trees worldwide, although the engineered vector form is derived from a less virulent strain, at least for Florida citrus trees (still highly virulent in California trees). Prior studies have purportedly demonstrated that CTV-based vectors can express engineered inserts in plant cells (U.S. Pat. No. 8,389,804; US 20100017911 A1). However, it has not been commercialized due to its inconsistent ability to accumulate in plants and achieve its targeted beneficial outcome. It is thought that CTV's inability to replicate to sufficiently high levels and heat sensitivity limits its ability to generate a sufficient quantity of RNA for treatment.

Thus, CTV-based vectors have a very limited ability to deliver an effective beneficial payload where needed. Moreover, CTV is difficult to work with due to its large size. CTV is also subject to superinfection exclusion, wherein a CTV-based vector is unable to infect a tree already infected with CTV. In addition, strains suitable for one region (e.g., Florida) are unsuitable for varieties of trees in another region (e.g., California). CTV also encodes three RNA silencing suppressors making its ability to generate large amounts of siRNAs problematic. Despite such problems, CTV is the only viral vector platform available for citrus trees.

Accordingly, there is a need for an alternative infectious agent, optionally that solves some or all of the above-noted problems, and which is capable of introducing a desirable property and/or delivering a therapeutic agent(s) into a plant, optionally for an extended period of time, particularly a long-lived plant such as a tree or vine.

Viral vectors may be derived from a wild-type virus and modified with an exogenous insert. The presence of some or all of the wild-type viral genome allows for replication of the vector, either in the host being treated or in manufacture outside of a host. The exogenous insert provides an active agent to achieve some form of activity from the vector. In some examples, the exogenous insert includes RNA that will be converted by the plant into a small interfering RNA (siRNA), which is typically 21-24 nucleotides (nt) in length. In one conventional method, the siRNA sequence is included in a base-paired, double-stranded (“hairpin”) structure. The siRNA sequence extends along one side of the hairpin, and a complementary base-paired sequence extends along the other side of the hairpin. The two sides of the hairpin are separated by an apical loop. Small hairpins are easier to work with, and so the size of the siRNA haipin is typically less than 60 nt.

Hairpins can be highly stable in a structural sense. For example, each base may have minimal or no positional entropy (PE), and each base pair may have a high probability of forming. However, fully base-paired hairpins, particularly hairpins with many G-C base pairs, can result in vectors with low stability for replication, i.e. the vectors are unable to maintain the inserts within their genome as they replicate. As the vector replicates, the hairpins may be deleted from some progeny of the vector. Progeny without the hairpin may be closer to the wild type vector. Since the wild type virus has evolved to be optimally fit, vector progeny without the hairpin may out compete vector progeny with the hairpin until eventually the hairpin in lost. Accordingly, while a viral vector with a hairpin can remain effective for a period of time, the stability of viral vectors may be further improved. Further, it is desirable to increase the size of the targeting sequence. Even when producing a VIGS vector, although the inserts are cut into segments of 21-24 nt in the plant it is beneficial to provide a longer targeting sequence that may be cut into more than one 21-24 nt sequence, or a targeting sequence complementary to multiple targets.

This specification describes RNA inserts for a vector that are hairpin-like, or include a hairpin-like region, but not are not conventional fully base-paired hairpins (i.e., the hairpin-like inserts are not fully base-paired outside of their apical loop). In at least some examples, these inserts carry larger targeting sequences and/or provide increased stability for replication than previously described inserts. Increased stability may involve a lower incidence of replicates that have deleted the insert at a given time point, or by replicates with the insert being detectable in the host for a longer period of time. The words stable and unstable may be used herein as relative terms. Inserts described as unstable may have some stability and could be useful for some applications. Hairpins that are described as stable are stable relative to other hairpins, but less stable than stable hairpin-like structures.

This specification also describes vectors that include an exogenous segment. In some examples, the exogenous segment is a hairpin-like structure having two or more base-paired regions, one or more non-base-paired regions separating the base-paired regions, and an apical loop at the end of one of the based paired regions. In some examples, the exogenous segment further complies with one or more design guidelines or parameters. These guidelines or parameters for the exogenous segment may include: the average positional entropy (APE) is in the range of 0.01 to 0.75; the length of the exogenous segment is 300 nt or less; the maximum length of a base-paired region is 19 base pairs; the maximum APE of a base-paired region is 0.8; the maximum number of consecutive G:C pairs in a base-paired region is 4; the maximum number of bases in a non-base-paired region is 20; the ΔG of the exogenous segment is within a range of −5 to +15 kcal/mol or within 10 kcal/mol (+ or −) of the ΔG of a naturally occurring hairpin of similar length; the standard deviation of PE is less than 0.5, less than 15% of bases have a PE greater than 1; the largest PE of any base is not greater than 2.0; and the insert, not considering the apical loop, is 65-90% base-paired. Although inserts are preferably hairpin-like structures, optionally a hairpin may be designed according to one or more of the design guidelines or parameters described herein.

In some examples, a vector includes an exogenous segment that has been designed to mimic a model hairpin-like structure in a wild type virus. In some examples, the exogenous segment mimics the secondary structure of the wild type hairpin-like structure by having a similar arrangement of base-paired regions and non-base-paired region. In some examples, the exogenous segment mimics the secondary structure of the wild-type hairpin-like structure by having a similar ΔG relative to length. Optionally, the vector is derived from the wild type virus having the model hairpin-like structure, or one or more relatives of that wild type virus. A mimicked exogenous segment may also follow one or more of the guidelines or parameters described above. Alternatively, a mimicked exogenous segment may follow one or more analogous guidelines or pararameters derived from a study of hairpin-like structures in a wild type virus that the vector is derived from, or one or more relatives of that wild type virus.

In the detailed description, we describe the complete secondary structure of citrus yellow vein associated virus (CYVaV) and some or all of the secondary structure of some of its related umbravirus-like associated RNA (ulaRNA). CYVaV and other ulaRNA are able to replicate and move systemically in plants, in many cases with minimal or no symptoms of infection. CYVaV and other ulaRNA can be developed into vectors to control vascular diseases in trees and vines and/or to target plant pathogens such as insects, fungi and other viruses. In some examples, a vector is derived from a ulaRNA, a Class 2 ulaRNA or CYVaV. These vectors may have exogenous inserts that comply with one or more design guidelines or parameters described above, or mimick a naturally occurring hairpin-like structure in a ulaRNA, a Class 2 ulaRNA or CYVaV. Although the detailed description is focused on ulaRNA, the invention is not limited to ulaRNA.

The present disclosure is also related to a modified vector, for example r a virus induced gene silencing (VIGS) vector. The vector is modified by the addition of a heterologous element that comprises one or more nucleotide sequences not found in the wild-type virus (exogenous segments). The exogenous segment(s) may substantially mimic the secondary structure of a portion, for example a hairpin-like portion, of the wild type vector (e.g. virus), or of a relative of the wild type vector. A hairpin-like portion and its corresponding exogenous segment may each comprise two or more base-paired regions and one or more non-base-paired regions. In some examples, a hairpin-like portion and exogenous segment each comprise one or more non-base-paired regions each located between two base-paired regions. In some examples, the non-base-paired region may be single sided or double sided and form a “loop” or “bulge” between two base-paired regions in the secondary structure of the vector. In some examples, the exogenous segment may have a sequence with many, for example 60 or more, 70 or more, 80 or more or 100 or more, bases, or changes in bases relative to the wild type vector. The exogenous segment may thereby contain an active (i.e. targeted) sequence that is 28 nt or more, 30 nt or more, 35 nt or more, 40 nt or more, or 50 nt or more, in length. In some examples, the exogenous segment contains at least one non-base-paired region. In some examples, an exogenous segment has a minimum free energy similar to a double stranded or hairpin-like portion of the wild type vector. In some examples, an exogenous segment has an active portion, for example an siRNA targeted to a pathogen or a gene silencing RNA targeted at a host plant.

In some examples, an exogenous segment is inserted into a vector a) in the location of, and as a replacement for, a hairpin-like structure that the exogenous segment mimics, b) in the location of, and as a replacement for, a hairpin-like structure that the exogenous segment does not mimic (wherein the exogenous segment mimics a different hairpin-like structure optionally of the vector or a relative of the vector), or c) at a location that previously did not have any hairpin-like structure (wherein the exogenous segment mimics a hairpin-like structure, optionally of the vector or a relative of the vector).

The present disclosure also relates to a novel infectious agent(s) capable of delivering and stably maintaining an exogenous insert(s) into a plant, compositions comprising a plant infected by the disclosed agent(s), and methods and uses relating thereto. The disclosed agents are sometimes referred to herein as “independently mobile RNAs” or “iRNAs.” Despite being infectious single-stranded RNAs, some iRNAs do not encode for any movement protein(s). They also do not encode RNA silencing suppressors, which are a key characteristic of plant viruses. In addition, unlike virtually all plant RNA viruses, with the exception of umbraviruses, and contrary to some definitions of a virus, iRNAs also do not encode a coat protein for encapsidating the RNA into virions, which is a requirement for vectored movement of viruses from plant to plant. Despite the lack of movement protein expression in some examples, iRNAs are able to move systemically within the phloem in a host plant. As compared to viruses, iRNAs have additional advantageous properties, such as: the ability to accumulate to levels exceeding those of most known plant viruses; possessing a relatively small size, e.g., being only about two-thirds the size of the smallest plant RNA virus and thus much easier to work with compared to such conventional plant RNA viruses; and exhibiting the inability to spread on their own to other plants (given their inability to encode for any coat protein). The lack of a silencing suppressor allows the immune system of the host plant to slice or break up the iRNA, thereby releasing siRNAs from the exogenous inserts of the iRNA into the plant. iRNA include umbravirus-like associated RNA (ulaRNA).

In accordance with disclosed embodiments, an infectious agent comprises an RNA, e.g. an iRNA, which may contain one or more engineered insert(s), sometimes referred to herein as a heterologous segment(s) or alternatively as exogenous segment(s), which, for example, triggers in a plant expression of a targeted peptide, protein(s) and/or produces targeted siRNA or other non-coding RNA that are cleaved from the vector for beneficial application, and/or delivers a therapeutic agent into the plant, and/or otherwise effectuates or promotes via such targeting or delivery a beneficial or desired result. Aspects of the present disclosure include: an iRNA-based vector for delivery of targeted anti-pathogenic agents; an anti-bacterial enzybiotic targeted at bacteria infecting a plant or bacteria required by the insect vector; an enzybiotic that is generated from an internal ribosome entry site (IRES) of the tobacco etch virus (TEV) (TEV IRES); incorporation of siRNAs into the iRNA genome; incorporation of inserts into a lock and dock structure to stabilize the base of a scaffold that supports the inserts; incorporation of sequences that may be cut into siRNAs into an iRNA genome that has been modified to enhance the structural stability of the local region to counter the destabilizing effects of the inserts; incorporation of an siRNA that disrupts or kills a targeted insect vector; incorporation of an siRNA that mitigates the negative impacts of a tree's callose production; incorporation of an siRNA that mitigates the plant's recognition of the pathogen; incorporation of an siRNA or other agent that targets bacterial, viral or fungal pathogens; and incorporation of an insert that triggers a particular plant trait (e.g., dwarfism). Thus, the infectious agents and compositions disclosed herein possess superior and advantageous properties as compared to conventional technologies.

The iRNA-based vectors of the present disclosure are suitable for use as a general platform for expression of various proteins and/or delivery of small RNAs into the phloem of citrus and other host plants. In some implementations, a citrus yellow vein associated virus (CYVaV)-based vector is provided, which accumulates to massive levels in companion cells and phloem parenchyma cells. The vectors of the present disclosure may be utilized to examine the effects of silencing specific gene expression, e.g., in the phloem (and beyond) of trees. In addition, CYVaV may be developed into a model system for examining long-distance movement of mRNAs through sieve elements. Since CYVaV is capable of infecting virtually all varieties of citrus, with few if any symptoms generated in the infected plants, movement of RNAs within woody plants may be readily examined.

In accordance with disclosed embodiments, the present disclosure is directed to a plus-sense single stranded ribonucleic acid (RNA) vector comprising a replication element(s) and a heterologous segment(s), wherein the RNA vector lacks a functional coat protein(s) open reading frame(s) (ORFs) and optionally lacks a functional movement protein ORF. The RNA vector is capable of movement in a host plant, for example systemic movement, movement through the phloem, long-distance movement and/or movement from one leaf to another leaf. In some implementations, the RNA vector also lacks any silencing suppressor ORF(s). In some implementations, the RNA vector comprises a 3′ Cap Independent Translation Enhancer (3′ CITE) comprising the nucleic acid sequence(s) of SEQ ID NO:4 and/or SEQ ID NO:5. In some embodiments, the 3′ CITE comprises the nucleic acid sequence of SEQ ID NO:3.

In some embodiments, the replication element(s) of the RNA vector comprises one or more conserved polynucleotide sequence(s) having the nucleic acid sequence of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and/or SEQ ID NO:14. In some implementations, the replication element(s) additionally or alternatively comprises one of more conserved polynucleotide sequence(s) having the nucleic acid sequence of SEQ ID NO:15 and/or SEQ ID NO:16.

In some embodiments, the RNA vector is derived from citrus yellow vein associated virus (CYVaV) (SEQ ID NO:1) or an iRNA or ulaRNA relative thereof, for example a Class 2 ulaRNA. The RNA vectors of the present disclosure are capable of systemic and phloem-limited movement and replication within a host plant. The RNA vectors of the present disclosure are functionally stable for replication, movement and/or translation within the host plant for at least one month after infection thereof, more preferably for at least 3 months, at least 6 months, at least 12 months, or at least 2 years, after infection thereof. In preferred embodiments, the RNA vectors and inserts thereof are functionally stable for the life of the host plant (e.g. 5-10 years or more).

In some embodiments, the heterologous segment(s) of the RNA vector of the present disclosure comprises a polynucleotide that encodes at least one polypeptide selected from the group consisting of a reporter molecule, a peptide, and a protein or is an interfering RNA. In some implementations, the polypeptide is an insecticide or an insect control agent, an antibacterial, an antiviral, or an antifungal. In some implementations, the antibacterial is an enzybiotic. In some implementations, the antibacterial targets a bacterium Candidatusspecies, e.g. Candidatus(CLas).

In some embodiments, the heterologous segment(s) of the RNA vector of the present disclosure comprises a small non-coding RNA molecule and/or an RNA interfering molecule. In some implementations, the small non-coding RNA molecule and/or the RNA interfering molecule targets an insect, a bacterium, a virus, or a fungus. In some implementations, the small non-coding RNA molecule and/or the RNA interfering molecule targets a nucleic acid of the insect, the bacterium, the virus, or the fungus. In some implementations, the small non-coding RNA molecule and/or the RNA interfering molecule targets a virus, for example a virus selected from the group consisting of Citrus vein enation virus (CVEV) and Citrus tristeza virus (CTV). In some implementations, a targeted bacteria is(CLas). In some implementations, the iRNA comprises an siRNA hairpin or hairpin-like structure that targets and renders the targeted bacteria non-pathogenic.

It should be understood that the RNA vector may include multiple heterologous segments, each providing for the same or different functionality. In some embodiments, the heterologous segment(s) is a first heterologous segment, wherein the RNA vector further comprising a second heterologous segment(s), wherein the replication element(s) is intermediate the first and second heterologous segments.

In some embodiments, the heterologous segment(s) of the RNA vector of the present disclosure comprises a polynucleotide that encodes for a protein or peptide that alters a phenotypic trait. In some implementations, the phenotypic trait is selected from the group consisting of pesticide tolerance, herbicide tolerance, insect resistance, reduced callose production, increased growth rate, and dwarfism.

The present disclosure is also directed to a host plant comprising the RNA vector of the present disclosure. The host plant may be a whole plant, a plant organ, a plant tissue, or a plant cell. In some implementations, the host plant is in a genus selected from the group consisting of citrus,and. In some implementations, the host plant is a citrus tree or a citrus tree graft.

The present disclosure also relates to a composition comprising a plant, a plant organ, a plant tissue, or a plant cell infected with the RNA vector of the present disclosure. In some implementations, the plant is in a genus selected from the group consisting of citrus,, and. In some implementations, the plant is a citrus tree or a citrus tree graft.

The present disclosure also relates to a method for introducing a heterologous segment(s) into a host plant comprising introducing into the host plant the RNA vector of the present disclosure. In some embodiments, the step of introducing the heterologous segment(s) into the host plant comprises grafting a plant organ or plant tissue of a plant that comprises the RNA vector of the present disclosure to a plant organ or plant tissue of another plant that does not comprise the RNA vector prior to said introduction. The RNA vectors of the present disclosure are capable of systemically infecting the host plant.

The present disclosure is also directed to a process of producing in a plant, a plant organ, a plant tissue, or a plant cell a heterologous segment(s), comprising introducing into said plant, said plant organ, said plant tissue or said plant cell the RNA vector of the present disclosure. In some embodiments, the plant is in a genus selected from the group consisting ofand

The present disclosure also relates to a kit comprising the RNA vector of the present disclosure.

The present disclosure is also directed to use of the RNA vector(s) of the present disclosure for introducing the heterologous segment(s) into a plant, a plant organ, a plant tissue, or a plant cell. The present disclosure is also directed to use of the host plant(s) of the present disclosure, or use of the composition(s) of the present disclosure, for introducing the RNA vector(s) into a plant organ or plant tissue that does not, prior to said introducing, comprise the RNA vector. In some implementations, the step of introducing the RNA vector comprises grafting a plant organ or plant tissue of a plant that comprises the RNA vector to a plant organ or plant tissue of another plant that does not comprise the RNA vector.

The present disclosure is also directed to a method of making a vector for use with a plant comprising the steps of inserting one or more heterologous segment(s) into an RNA, wherein the RNA is selected from the group consisting of: CYVaV; a relative of CYVaV; other RNA vectors having least 50% or at least 70% RdRp identity with CYVaV; and another iRNA or ulaRNA. The present disclosure also relates to a vector produced by the disclosed method(s).

The present disclosure also relates to the use of an RNA molecule as a vector, wherein the RNA is selected from the group consisting of: CYVaV; a relative of CYVaV; other RNA vectors having at least 50% or at least 70% RdRp identity with CYVaV; and, another iRNA or ulaRNA. In some implementations, the RNA is used in the treatment of a plant, for example the treatment of a viral or bacterial infection of a plant, for example the treatment of CTV infection or Citrus Greening in a citrus plant, or in the control of insects that are vectors and/or feed on the plant. The RNA is modified with one or more inserted heterologous segment(s), for example an enzybiotic or an siRNA.

The present disclosure is also directed to the use of an RNA molecule characterized by being in the manufacture of a medicament to treat a disease or condition of a plant, wherein the RNA is selected from the group consisting of: CYVaV; a relative of CYVaV; other RNA vectors having at least 50%, or at least 70%, identity with the RdRp of CYVaV; and, another iRNA or ulaRNA. In some implementations, the disease or condition is a viral or bacterial infection of a plant, for example CTV or Citrus Greening in a Citrus plant.

The present disclosure is also directed to an RNA molecule for use as a medicament or in the treatment of a disease or condition of a plant, wherein the RNA is selected from the group consisting of CYVaV; a relative of CYVaV; other RNA vectors having at least 50% or at least 70% RdRp identity with CYVaV; and, another iRNA or ulaRNA.

The present disclosure is also related to a ribonucleic acid (RNA) vector, for example a plus-sense single stranded ribonucleic acid (RNA) vector, comprising one or more heterologous segment(s), wherein said heterologous element(s) is attached to the main structure of the RNA vector through a lock and dock structure, optionally a branched structure comprising an insert site for the heterologous element and a relatively stable and/or locking structure that does not participate in folding of the heterologous element or the main structure of the RNA vector. In some implementations, the RNA vector is an iRNA or ulaRNA-based vector or a virus-based vector. In some implementations, a lock portion of the lock and dock structure comprises a scaffold normally used for crystallography. In some implementations, the lock and dock structure comprises a branched element, wherein a stem and a branch of the branched element are located within a relatively stable structure forming the lock, such as a tetraloop-tetraloop dock, e.g., a GNRA tetraloop docked into its docking sequence, and another branch of the branched element comprises an insert site for the heterologous element. In some implementations, the heterologous element is a hairpin or an unstructured sequence.

The present disclosure is also related to an iRNA or ulaRNA-based vector having one or more heterologous segment(s) having a sequence that targets a particular pathogen, e.g., such as a virus, a fungus, or a bacteria. In some implementations, the siRNA is effective against a plant pathogenic bacterium. In some implementations, the siRNA targets a Candidatusspecies such as Candidatus(CLas).

The present disclosure is also related to a vector having a heterologous element comprising a hairpin or hairpin-like structure having a sequence on one side complementary to a sequence within citrus tristeza virus (CTV) or an unstructured sequence complementary to the plus or minus strand of CTV. In some implementations, the sequence within CTV is conserved in multiple CTV strains. In some implementations, the sequence one on side of the hairpin or hairpin-like structure is complementary with a sequence in multiple CTV strains, or all known CTV strains, despite differences in CTV sequences. The present disclosure is also related to a plant having a sour orange rootstock and an iRNA or ulaRNA-based vector having a heterologous element that targets Citrus tristeza virus.

The present disclosure is also related to a method for introducing a heterologous segment(s) into a host plant comprising introducing into said host plant an iRNA or ulaRNA-based vector after a) encapsidating the iRNA or ulaRNA vector in a capsid protein other than the capsid protein of CVEV, or b) by coating the iRNA or ulaRNA with phloem protein 2 (PP2) from sap extracted from cucumber, citrus or other plant, c) by using dodder to take up sap from infected laboratory host and transmit to a secondary host, e) by encapcidating the iRNA or ulaRNA in virions of CVEV and infecting plants by stem slashing or stem peeling, or f) by feeding CYVaV-containing virions to a CVEV-specific aphid vector and then allowing the aphids to feed on trees.